The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.
Mammals are required to defend themselves against a multitude of pathogens including viruses, bacteria, fungi and parasites, as well as non-pathogenic insults such as tumours and toxic, or otherwise harmful, agents. In response, effector mechanisms have evolved which are capable of mounting a defence against such antigens. These mechanisms are mediated by soluble molecules and/or by cells.
In the context of these effector mechanisms, inflammation is a complex multifaceted process in response to disease or injury and which is regulated by the release of a series of cytokines (Alexander et al, 2001, J Endotoxin Res 7:167-202). These cytokines are classified in general terms as pro-inflammatory or anti-inflammatory cytokines and the critical balance between release and activity of cytokines with opposing actions regulates the inflammatory response to prevent it from becoming overt or understated.
If the inflammatory response continues unchecked and is overt then the host may suffer associated tissue damage and in severe cases this may present as septic shock and multi-organ failure can occur (Ulevitch et al., 1999, Curr Opin Immunol 11:19-22). Conversely, a poor or understated inflammatory response may mean uncontrolled infection resulting in chronic illness and host damage. Regulation of the inflammatory response is important at both the systemic level and the local level.
The discovery of the detailed processes of inflammation has revealed a close relationship between inflammation and the immune response. There are five basic indicators of inflammation, these being redness (rubor), swelling (tumour), heat (calor), pain (dolor) and deranged function (functio laesa). These indicators occur due to extravasation of plasma and infiltration of leukocytes into the site of inflammation. Consistent with these indicators, the main characteristics of the inflammatory response are therefore:    (i) vasodilation—widening of the blood vessels to increase the blood flow to the infected area;    (ii) increased vascular permeability—this allows diffusible components to enter the site;    (iii) cellular infiltration—this being the directed movement of inflammatory cells through the walls of blood vessels into the site of injury;    (iv) changes in biosynthetic, metabolic and catabolic profiles of many organs; and    (v) activation of cells of the immune system as well as of complex enzymatic systems of blood plasma.
The degree to which these characteristics occur is generally proportional to the severity of the injury and/or the extent of infection.
In the context of the unique microenvironment of the human female reproductive tract, the mucosal epithelium of the reproductive tract acts as a physical barrier and expresses immunological mediators that serve as the first line of attack against infection (Hickey et al, 2011, J Reprod Immunol 88:185-194; Kaushic, C. 2011, Am J Reprod Immunol 65:253-260. The vagina and ectocervix comprise multiple layers of usually non-keratinized stratified squamous epithelium, with the uppermost permeable stratum corneum layer lacking tight junctions (Blaskewicz, et al. 2011, Biol Reprod 85:97-104), and are in contact with vaginal microbiota and semen. The cervical transformation zone separating the endocervix and ectocervix is the most immunologically active site in the reproductive tract with a relative abundance of lymphocytes and antigen presenting cells (Pudney, et al. 2005. Biol Reprod 73:1253-1263). Additionally, the vagina/ectocervix and endocervix exhibit immunological functions by conferring tolerance to microbes, maintaining epithelial integrity and recruiting and supporting of immune cells (Fichorova, R. N., and D. J. Anderson. 1999. Biol Reprod 60:508-514). As the front line of the innate immune response, reproductive tract epithelial cells express Toll-like receptors (TLRs) (Kaushic, C. 2011. Supra; Wira, et al. 2005. Am J Reprod Immunol 53:65-76). They respond to pathogen associated molecular patterns (PAMPs) via their TLRs by secreting cytokines and chemokines, antimicrobial peptides (defensins and secretory leukocyte peptidase inhibitor; SLPI), antimicrobial enzymes (lactoferrin and lysozome), surfactant protein A and complement (Fichorova, R. N., and D. J. Anderson. 1999. Supra; Hickey, et al. 2011, Supra; Kaushic, C. 2011, Supra). Nevertheless, disruption in the equilibrium of the reproductive tract, leading to the onset of inflammation is experienced by many women. The consequences of disturbances in this microenvironment range from annoying irritation through to an increased susceptibility to serious infections, such as HIV. To this end, three of the main causes of vaginal inflammation are:    (i) Atrophic vaginitis—This is most likely to be the result of having had a hysterectomy or occurs in a post menopausal woman. Atrophic vaginitis is estimated to affect as many as 40% of women. This type of inflammation is brought about due to the lack of estrogen stimulation of the vaginal tissue which can lead to the formation of adhesions, painful intercourse and an increased susceptibility to infections, itching burning, stinging and a watery discharge.    (ii) Irritant vaginitis—Irritant vaginitis is caused by an irritant. For example, sensitivity to a chemical being used or an allergic reaction to something one has come into contact with. This can be caused by a number of chemicals or physical agents, including tampons, the chemicals in scented feminine hygiene products, of personal lubricant, or even the harshness of the chemicals in the detergent being used to wash ones undergarments.    (iii) Infectious vaginitis—Infectious vaginitis is any type of vaginitis caused by microorganism infection—fungal, bacterial, viral, parasitic or otherwise. These include gardeneralla (a bacterial infection), any type of virus, STDs (like chlamydia, gonorrhea, or trichomoniasis), and vaginal yeast infection.
Although Lactobacillus sp dominate the microbiota of the healthy female reproductive tract and produce lactic acid (both L and D isoforms) to a concentration of approximately 1% which acidifies the reproductive tract to pH<4, the role, if any, of Lactobacillus sp., amongst the many other flora present in the reproductive tract, in terms of contributing to the health of the reproductive tract, has not been understood. That is, the vaginal microbiome is complex.
In fact, whereas Lactobacillus sp. have been shown to be associated with anti-inflammatory activity in the gut, the studies of Mossop et al (2011, Obstet. Gynecol. 118:840-6) demonstrated that with human female reproductive tissue, lactic acid, this being the major metabolite produced by Lactobacillus sp., is pro-inflammatory at low pH. This finding is not unexpected when one considers that the human female reproductive tract is a unique microenvironment. Specifically, the vagina is a highly acidic environment, more so than other organs or tissue. In fact, the human female reproductive tract is significantly more acidic than even the reproductive tracts of other non-human primates. Still further, it is known that carboxylic acids, such as acetic acid, and short chain fatty acids produced by bacterial vaginosis-associated bacteria are pro-inflammatory (Mirmonsef et al 2012, Am J Rep Immunol 67: 391). Again, this contrasts to the situation in the gut where the same carboxylic acids and short chain fatty acids are shown to exhibit anti-inflammatory effects.
Although lactic acid is included in the formulations of a range of vaginal gels which are currently on the market, in some of these gels it is included merely as an excipient. That is, as a largely inactive substance used as a vehicle for the active ingredient in the gel, this usually being a microbicide. In fact, the lactic acid in these formulations is used at a concentration significantly higher than the physiological concentrations present in the human vagina. Nevertheless, lactic acid is known to conveniently function as a preservative. Still further, since it is a naturally acid molecule, in some gels, although not used strictly as an excipient, it is included for its role in acid buffering in the presence of semen to kill sperm.
In fact, Acidform, an acid-buffering product, which inactivates spermatozoa, is formulated with 2% lactic acid and other acids such as citric, benzoic and alginic acid. Interestingly, a Phase I Randomized Safety Study investigating Acidform as a non-hormonal topical contraceptive demonstrated an increase in irritation in treated subjects, as well as a decrease in anti-inflammatory cytokine IL-1RA (Keller et al 2012, Plos ONE 7:e46901).
Similarly, this ability to reduce pH and thereby maintain a pH conducive to microbicidal activity is a useful adjunct to the major active components present in these gels.
In work leading up to the present invention, it has been surprisingly determined that although carboxylic acids have been shown to be anti-inflammatory in the gut and pro-inflammatory in the human reproductive tract, lactic acid functions entirely contrarily. Specifically, it has been unexpectedly found that within the human female reproductive tract, lactic acid exerts anti-inflammatory activity. Still further, it has also been determined that, in fact, carboxylic acids such as acetic acid and citric acid (which are often formulated together with lactic acid in vaginal gels), could actually antagonise the activity of lactic acid. Still further, this anti-inflammatory activity does not require the lactic acid to be used at high concentrations. Rather, use of lactic acid at physiological concentrations is anti-inflammatory.
The findings of the present invention have now facilitated the development of methods and agents directed to down-regulating inflammation in the female reproductive tract by administering lactic acid to the mucosal surface of the reproductive tract. Accordingly, there are now provided methods for both the therapeutic or prophylactic treatment of unwanted or inappropriate inflammatory responses of the female reproductive tract.